Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute in complex fashion to cell migration and signaling. We are addressing the following questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell video or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on functions of integrins and associated extracellular and intracellular molecules in the mechanisms and topographical regulation of cell migration. Cell and tissue dynamics occur in three-dimensional (3D) environments in vivo. We have established the importance of one-dimensional (1D) migration for understanding 3D migration. Using a newly developed procedure termed micro photoablation, we demonstrated that cell migration through aligned fibrillar 3D cell-derived matrices is readily mimicked by simple 1.5 micron-wide micropatterned lines in a process we term 1D migration. We showed that many aspects of 3D fibrillar cell migration, including spindle cell morphology, migration velocity (both increased in 1D and 3D compared to 2D), cytoskeletal organization (both actin- and microtubule-based), centrosome and Golgi orientation, and responses to contractile inhibitors are reproduced much more effectively by this system with 1D fibrillar topography versus the traditional two-dimensional surfaces used for cell culture. A particularly interesting finding involves differences in the cellular responses to inhibitors of contractility during cell migration under 1D, 2D, and 3D conditions. Treatment of fibroblasts with inhibitors of actomyosin contractility on 2D fibronectin-coated surfaces leads to a nominal increase in cell migration velocity and cell spreading, whereas the same treatment of cells plated on 1D (fibronectin-coated) or 3D fibrillar cell-derived matrix substantially inhibits migration greater than two-fold. Based on these observations, together with our finding that adhesions to the underlying substratum in 1D form a unique, lengthy adhesion structure unlike that found on 2D surfaces, we are testing whether changing the topography and physical structure of cell-ECM adhesions affects the basic morphological and biochemical mechanisms proposed to mediate cell migration. In order to quantify the dynamics of proteins potentially comprising a clutch-like mechanism implicated in cell-matrix interactions, we are currently analyzing fluorescence recovery after photobleaching (FRAP) of GFP-linked fusion proteins together with other live-cell imaging techniques (Spinning Disk and TIRF microscopy) to track the dynamics of the proteins involved in the postulated molecular clutch to determine how 1D ECM enhances cell migration. More generally, we feel that studying cells migrating in 1D will provide a powerful new tool for analyzing the molecular mechanisms of cell migration, because the components of the molecular machinery are arrayed linearly along the length of a steadily migrating cell that remains oriented in a single direction. Nonmuscle cellular myosins and actin are thought to play crucial roles in cell migration and in many developmental and wound repair processes, but the roles of the major myosin IIA gene were not clear. We and others recently published studies on the roles of the major myosin II genes, myosin IIA and IIB. We found that myosin IIA plays central roles in fibroblast and embryonic stem cell contractility, actin cytoskeletal organization, and organization of cell-matrix adhesions. Unexpectedly and in contradiction to the belief that myosin II molecules are essential for cell migration, we showed that myosin IIA is not required, and in fact it serves as a brake on migration in 2D cell culture. We also found strong cross-regulation between myosin IIA and microtubule dynamics that regulates Rac localization and cell migration. For technical reasons, it was necessary in our original study to use standard 2D culture systems for visualizing cytoskeletal dynamics and Rho GTPase functions. We had also previously established a key role for Rac in helping to regulate directional cell migration in such 2D settings. Two new projects are testing whether these principles of myosin II crosstalk and Rho family GTPase functions in migration are correct in 1D, 2D, and 3D cell migration systems. These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosin and microtubules. All of these processes will need to be analyzed in parallel in real time and in more physiological 1D and 3D matrix environments to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.